The invention relates to human procalcitonin and the preparation, in particular by genetic engineering processes, and use thereof.
Procalcitonin (xe2x80x9cpCTxe2x80x9d) is a protein consisting of 116 amino acids and having a molecular weight of about 13,000 dalton. It is the prohormone of calcitonin which under normal metabolic conditions is produced and secreted by the C cells of the thyroid. pCT and calcitonin synthesis is initiated by translation of preprocalcitonin (xe2x80x9cpre-pCTxe2x80x9d), a precursor peptide comprising 141 amino acids. The amino acid sequence of human pre-pCT was described by Moullec et al. in FEBS Letters, 167:93-97 in 1984. pCT is formed after cleavage of the signal peptide (first 25 amino acids of pre-pCT). In healthy people the hormone calcitonin (amino acids 60-91 of the pCT amino acid sequence), and N-procalcitonin (amino acids 1-57 of the pCT amino acid sequence) and katacalcin (amino acids 96-116 of the pCT amino acid sequence) are produced intracellularly from pCT by specific proteolysis (see also Conlan et al. (1988) Biochem. J., 256:245-250). pCT and fragments thereof were detected in increased concentrations in the serum or plasma of patients, in particular in cases of certain neoplastic diseases (Ghillani et al. (1989) Cancer Research, 49:6845-6851) and sepsis (EP-B1-0 656 121) and SIRS (systemic inflammatory response syndrome) (Snider et al., (1997) J. Investig. Med., 45:552-560).
During the typical sepsis bacteria are released continuously or in phases from a focus into the bloodstream. Endotoxin or other pyrogenic and toxic substances interacting with body mechanisms cause the clinical manifestations. The acute onset triggers chills and in severe cases a shock reaction. Special forms of septic shock are Waterhouse-Friderichsen syndrome and toxic shock syndrome (TSS). TSS is known as an acute clinical picture in staphylococcal infections which is caused by a specific staphylococcal toxin. A severe sepsis quite frequently develops in patients with serious primary disorders such as, for example, neoplastic diseases, serious burns and traumas.
The importance for sepsis diagnosis of detecting pathogens in the blood (xe2x80x9cpositive blood culture, bacteremiaxe2x80x9d) has been pushed into the background, because in general the blood culture is positive only in 20 to 40% of sepsis cases. The term sepsis has therefore undergone a change. The modern term xe2x80x9csepsisxe2x80x9d describes a clinical syndrome which in general comprises fever, leukocytosis, alterations of consciousness, a hyperdynamic circulation (xe2x80x9cwarm shockxe2x80x9d) and a hypermetabolic state, a positive blood culture no longer being required as a prerequisite for sepsis diagnosis.
WO 98/33524 suggests employing antibodies binding to pCT for the therapy of sepsis and SIRS.
Over many years polyclonal antibodies were obtained from immunization by calcitonin and used for detecting so-called immunoreactive calcitonin which aside from calcitonin also comprises procalcitonin and further procalcitonin fragments. Immunization by synthetic peptides having amino acid sequences corresponding to the sequences of procalcitonin segments succeeded in producing various monoclonal antibodies binding to various calcitonin and katacalcin epitopes (Ghillani et al. (1988) J. Immunol., 141:3156-3163).
On the basis of these antibodies sandwich immunoassays for detecting pCT and calcitonin in serum samples were also developed. A combination of an anti-katacalcin antibody and an anti-calcitonin antibody was suggested for detecting calcitonin precursor molecules. A synthetic peptide suited to these antibodies was employed as standard material.
It is known that in immunochemical tests the measured signals for standards and samples need not necessarily be identical even if the amount of antigen is exactly the same. If standard and sample antigens are not really identical regarding their immunochemical reactivity, the antibodies employed in the test will recognize either the one or the other antigen better. This leads in the end to different measured signals for samples and standards.
It follows from this that the use as standard antigen of antigen fragments instead of the whole protein is often associated with disadvantages and can, in particular, lead to distorted measurements. Furthermore, it is in general not possible for the epitopes based on the three-dimensional structure of the correctly folded protein to be correctly represented by shorter peptides. This results in it not being possible to obtain antibodies against such conformation epitopes on use of peptides as immunogens. It is advantageous especially in competitive test formats if the substance to be detected has the same immunochemical reactivity as the corresponding solid phase or label-bound test reagent.
Although the complete amino acid sequence of human pCT has now been known since 1984, so far human pCT has not been prepared successfully, in particular not in relatively large amounts and reproducibly. So far only murine pCT could be expressed in E. coli by means of genetic engineering processes (Rehli et al. (1996) Biochem. Biophys. Res. Com., 226:420-425).
However, murine pCT differs from human pCT to such an extent (about 77% homology at the amino acid level) that it is still an object for the skilled worker to develop a process by which human pCT can be produced in relatively large amounts, cost effectively and in isolated form in order to be able to employ it particularly as an immunogen and/or standard and control sera antigen.
This object is achieved by providing the polypeptides according to the invention described in claims 1-3, the plasmids according to the invention described in claims 10 and 11, the cells according to the invention described in claim 12 and the preparation processes according to the invention described in claims 4-9. The polypeptides according to the invention, i.e. the polypeptides as claimed in one of claims 1-3 or the products of a process as claimed in claims 4-9, can be employed usefully in particular in the fields of diagnosis and therapy. Preferred embodiments of the invention are disclosed in claims 13-23. Furthermore, the polypeptides according to the invention can be used for immunization to obtain the antibodies according to the invention. A further embodiment of the invention is the pCT solutions described in claims 24-29.
It was not possible to predict the feasibility of human pCT expression according to the invention: pCT is expressed in the cell not as pCT, but originates from preprocalcitonin by proteolytic cleavage of the N-terminal signal peptide. It had to be assumed that in eukaryotes pCT without signal peptide is not expressed in the natural cell compartment and folds in a different way, potentially causing biological inactivation and possibly even instability. In addition, the heterologous expression in E. coli instead of the natural expression in animal cells and the expression attempted within the framework of the invention of a fusion protein of procalcitonin and the artificial sequence MRSHHHHHHGS (part of SEQ ID NO: 9) N-terminally therefrom could not be foreseen as prospectively successful. Although expression in E. coli of murine pCT as a poly-His fusion protein has been described in the literature (Rehli et al.), no conclusions can be drawn from this for the feasibility of human pCT expression since there is only about 77% identity at the amino acid level and therefore a completely different behavior must be expected. Furthermore, the murine pCT was not expressed directly after the putative signal peptide cleavage site (A25/V26) (Jakobs et al., 1981, Science, 213: 457-457), but only a murine procalcitonin fragment shortened by 7 amino acids was expressed, which again may have unforeseeable consequences for the expressibility. Finally, the publication contains neither the exact fermentation conditions nor the achievable yields after purification of the fusion protein.
In the following specific embodiments of the invention are described in more detail:
The invention relates preferably to an isolated polypeptide comprising the amino acid sequence of human pCT, in particular if prepared using genetic engineering processes. The amino acid sequence of human pCT is shown in FIG. 1 (SEQ ID NO: 7).
The term xe2x80x9cgenetic engineering processesxe2x80x9d in accordance with this invention also means in particular processes in which the polypeptide to be expressed is produced by eukaryotic or prokaryotic cells, the nucleic acid sequence coding for the polypeptide to be expressed and including recombinant nucleic acid sequences being introduced previously into these cells, for example by means of vectors, liposomes, projectiles or co-precipitation with salts. In another process a gene already naturally present in the cell and coding for the polypeptide to be expressed is activated by activating measures, for example by gene amplification or activation by means of artificially introduced promoter and/or enhancer sequences or deletion of repressor binding sequences such that the cell expresses the polypeptide to be expressed in larger amounts than naturally.
The term xe2x80x9camino acid sequence of human pCTxe2x80x9d in accordance with this invention also means amino acid sequences slightly altered by exchange, deletion or addition of amino acids, and these alterations should have no serious negative influence on the binding properties of the pqlypeptide towards anti-pCT antibodies. The skilled worker can check this on the basis of appropriate binding studies using available anti-pCT antibodies.
The term xe2x80x9cpeptidesxe2x80x9d in accordance with this invention comprises amides which decompose into amino acids on hydrolysis, for example amino acid polymers such as, for example, polypeptides, oligopeptides, proteins or protein fragments. Molecules with no more than ten linked amino acids are in general called oligopeptides, with more than that they are called polypeptides.
Further polypeptides according to the invention are isolated polypeptides which contain the amino acid sequence shown in FIGS. 2A or 2B and which have preferably been prepared using genetic engineering processes.
A preferred process according to the invention is a process for the preparation of human procalcitonin wherein (i) a gene coding for a polypeptide comprising the amino acid sequence of human procalcitonin is inserted into a vector, (ii) a host organism is transformed with this gene-containing vector and (iii) the polypeptide expressed by the host organism is isolated. Preferred variant of this process according to the invention are those preparing a polypeptide having the amino acid sequence according to FIGS. 1 (SEQ ID NO: 7), 2A (SEQ ID NO: 8) or 2B (SEQ ID NO: 9).
A xe2x80x9cvectorxe2x80x9d is in particular a DNA or RNA molecule which is capable of replication in a host organism and from which a recombinant DNA or RNA molecule can be constructed by incorporation of one or more foreign genes. Examples of common vectors are bacterial plasmids; viral genomes, in particular genomes of bacteriophages; yeast chromosomes and plasmids, in particular YEp, YIp, YRp, YAC; Ti plasmid; and vectors derived from adenoviruses, papillomaviruses and retroviruses. In order to facilitate expression of a foreign gene a vector in general has a promoter which is, if possible, physically or chemically inducible and which initiates transcription of a messenger RNA coding for the protein to be expressed. Furthermore a vector in general has a nucleic acid sequence causing a transcription stop and nucleic acid sequences causing a very efficient translation such as, for example, a ribosome binding site in the case of bacterial expression. Additionally an expression vector should have translation stop sequences in all possible reading frames.
Particularly preferred processes according to the invention comprise the vector, for example pQE-30 (SEQ ID NO: 10), coding for a fusion segment, preferably polyhistidine, which later on permits a simple purification of the procalcitonin fusion protein.
Suitable host cells for the process according to the invention are human, animal, plant or prokaryotic cells; particularly preferred are E. coli cells.
A xe2x80x9cfusion proteinxe2x80x9d in accordance with this invention means a protein which comprises pCT and a further either C- or N-terminal poly- or oligopeptide which is translated in its entirety as one polypeptide. The fusion segment preferably ought to either increase the expressibility of procalcitonin and/or facilitate a later simple purification by affinity chromatography of the fusion protein.
Preferably the processes according to the invention will employ metal affinity chromatography and/or gel filtration to isolate the polypeptide according to the invention.
Metal affinity chromatography makes use of the fact that a chromatography gel matrix containing chelated doubly charged metal ions, for example Ni2+, which still have a plurality of freely accessible coordination sites can bind reversibly to proteins containing a plurality of histidines in succession. Elution of the polyhistidine polypeptide under mild conditions may then be achieved competitively, for example by imidazole-containing buffers.
This invention further relates to a plasmid containing one or more nucleic acid sequences coding for one or more of the polypeptides according to the invention. A very particularly preferred plasmid according to the invention named internally PQE-PCT was deposited under deposition number DSM 13203 at the DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, Braunschweig, Germany on Dec. 16, 1999.
Further embodiments according to the invention are animal, plant, isolated human or prokaryotic cells which can express one or more polypeptides according to the invention.
Yet another embodiment of this invention is the use of the polypeptides according to the invention as immunogens for preparing antibodies. The process according to the invention for preparing antibodies comprises the use as immunization antigen of one or more polypeptides according to the invention. Antibodies obtained by means of these process are called xe2x80x9cantibodies according to the inventionxe2x80x9d hereinafter.
The term xe2x80x9cantibodyxe2x80x9d in accordance with this invention means an immunoglobulin, for example an immunoglobulin of the class or subclass IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM. An antibody comprises at least one binding site often called paratope) for an epitope (often also called antigenic determinant) on an antigen or hapten. Such an epitope is characterized, for example, by its three-dimensional structure and/or the presence of polar and/or apolar groups. The antibody binding site is complementary to the epitope. The antibody binding site is complementary to the epitope. The antigen-antibody reaction or hapten-antibody reaction works according to the so-called xe2x80x9clock-and-key principlexe2x80x9d and is in general specific to a high degree, i.e. the antibodies are capable of distinguishing small difference in primary structure, charger, three-dimensional configuration and steric arrangement of the antigen or hapten. In particular the so-called complementarity determining regions of the antibody contribute to the binding of the antibody to the antigen of hapten.
The term xe2x80x9cantigensxe2x80x9d comprises monovalent and polyvalent antigens. A polyvalent antigen is a molecule or a molecule complex to which more than one immunoglobulin can bind simultaneously, whereas only a single antibody can bind to a monovalent antigen at any one time. A hapten usually denotes a molecule which on its own is not immunogenic but is usually bound to a carrier for immunization purposes.
The term antibody in accordance with this invention means not only complete antibodies but expressly also antibody fragments such as, for example, Fab, Fv, F(abxe2x80x2)2, Fabxe2x80x2; and also chimeric, humanized, bi- or oligo-specific or single-chain antibodies; furthermore also aggregates, polymers and conjugates of immunoglobulins and/or fragments thereof as long as the binding properties to the antigen or hapten are maintained. Antibody fragments can be prepared for example by enzymatic cleavage of antibodies using enzymes such as pepsin or papain. Antibody aggregates, polymers and conjugates can be generated by a multiplicity of methods, for example by heat treatment, reaction with substances such as glutaraldehyde, reaction with immunoglobulin-binding molecules, biotinylation of antibodies and subsequent reaction with streptavidin or avidin, etc.
An antibody according to this invention can be a monoclonal or a polyclonal antibody. The antibody may have been prepared according to the usual processes, for example by immunization of man or an animal such as, for example, mouse, rat, guinea pig, rabbit, horse, sheep, goat, chicken (see also Messerschmid (1996) BIOforum, 11:500-502) and subsequent preparation of the antiserum; or by establishing hybridoma cells and subsequent purification of the secreted antibodies; or by cloning and expression of nucleotide sequences or modified versions thereof coding for amino acid sequences which are responsible for binding of the natural antibody to the antigen and/or hapten.
In a preferred embodiment of the process according to the invention the polypeptides according to the invention which are used as immunization antigens may be used for the immunization in unbound and/or carrier-bound form. Typical carriers are, for example, proteins such as, for example, ovalbumin, albumin or hemocyanin, or polymers such as, for example, polyethylene glycol, polyacrylamide or poly-d-glutamine-d-lysine. The polypeptides can be bound to this carrier, for example, using carbodiimide or glutaraldehyde or else a bifunctional reagent which can also act as a spacer (for examples and coupling methods see e.g. Wong S. (1993) Chemistry of Protein Conjugation and Cross-linking, CRC Press Inc., Boca Raton).
The immunization antigen, for example, may be suspended in phosphate-buffered saline and treated with Freund""s adjuvant. This emulsion may then be administered, for example, intradermally, intraperitoneally and/or subcutaneously to an animal, for example a rabbit, mouse, rat, guinea pig, horse, sheep, goat, chicken, etc. Booster injections may help to increase the immune response, it also being possible for the immunization antigen to be emulsified with incomplete Freund""s adjuvant.
Polyclonal antibodies according to the invention may be obtained from the antiserum of the immunized animals. These antibodies can be further purified by means of affinity chromatography on a matrix to which, for example, pCT or pCT fragments have been bound.
In order to create monoclonal antibodies according to the invention the immune cells of immunized animals such as, for example, a mouse, are fused according to generally well-known methods (see e.g. Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor; Peters et al. (1985) Monoklonale Antikxc3x6rper: Herstellung und Charakterisierung, Springer Verlag) with myeloma cells to create hybridoma cells producing monoclonal antibodies (MAb), and subsequently suitable clones are isolated. The desired MAb-producing clones are selected using specific screening methods. In these the binding specificity of the antibodies released into the cell culture supernatant, for example for the immunization antigen, a possible carrier of the immunization antigen, pCT, free calcitonin, free katacalcin and free N-procalcitonin, is tested using, for example, enzyme immunoassays, radioimmunoassays and/or Western blots. Hybridomas producing the antibodies according to the invention are cloned. The hybridoma cell lines obtained in this way are then available for continuous MAb production. Larger quantities of antibodies may be obtained from, for example, cell culture supernatant, in particular from fermenters or roller cultures and from ascites.
It is advantageous depending on the desired purpose to employ only antibody fragments such as, for example, Fab, F(abxe2x80x2)2 or Fabxe2x80x2 fragments. These may be created, for example, by enzymatic cleavage methods known to the skilled worker (see also e.g. Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor).
The antigen binding sites of an antibody are located in the so-called variable domains coded for by the V genes. Using well-known genetic engineering methods (see e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 2nd edition; McCafferty et al. (1990) Nature 348:552-554) it is also possible to determine the corresponding nucleic acid sequence of an antibody according to the invention and thereby also the corresponding amino acid sequence, if not already known from amino acid sequencing. For analyses of this kind, the hybridoma cells or the antibody producing immune cells of immunized animals may be employed as starting material.
Knowing the nucleic acid sequence and/or amino acid sequence it is then possible using conventional genetic engineering and molecular biology methods (see also Johnson and Chiswell (1993) Current Opinion in Structural Biology, 3:564-571) to prepare humanized, chimeric, bi- or oligo-specific antibodies and peptides derived from the complementarity determining region (minimal recognition units), single-chain fragments and/or functional fusion products, for example recombinant antibody-enzyme constructs (see e.g. Larrick and Fry (1991) Human Antibodies and Hybridomas, 2:172-189; Kitano et al. (1986) Appl. Microbiol. Biotechnol., 24:282-286; Thompson et al. (1986) J. Immunol. Methods, 94:7-12) which bind to procalcitonin but not to free calcitonin, free katacalcin and free N-procalcitonin. Using such peptides included in the term xe2x80x9cantibodyxe2x80x9d it is possible, for example, to achieve a decrease in immunogenicity and/or an enhanced efficiency when administered as a medicament or in vivo diagnostic agent and/or there will be advantages when employed as or as part of an in vitro diagnostic agent. The antibodies may also be prepared using, where appropriate, genetic engineering methods in plant cells such as, for example, yeast cells (Fischer et al. (1999) Biol. Chem., 380:825-839; Hiatt et al. (1992) Genetic Engineering, 14:49-64)), animal and prokaryotic cells (see e.g. WO 95/25172) and isolated human cells.
Furthermore, this invention also relates to animal, plant or prokaryotic cells and isolated human cells producing an antibody according to the invention.
It is also possible for the skilled worker by providing the antibodies according to the invention to identify, for example by competition experiments (see also Peters et al. (1985) Monoklonale Antikxc3x6rper, Springer Verlag, chapter 12.2 xe2x80x9cEpitop-Analysexe2x80x9d), other specific binding partners expressly including antibodies which bind to the epitope of an antibody according to the invention. Thus it is possible now by techniques known to the skilled worker to select specific binding partners using phage display libraries, synthetic peptide databases or recombinatorial antibody libraries (Larrick and Fry (1991) Human Antibodies and Hybridomas, 2:172-189).
A xe2x80x9cspecific binding partnerxe2x80x9d means a member of a specific binding pair. The members of a specific binding pair are two molecules each having at least one structure complementary to a structure of the other molecule, the two molecules being able to bind to each other via binding of the complementary structures. The term molecule also comprises molecule complexes such as, for example, enzymes comprising apoenzyme and coenzyme, proteins comprising a plurality of subunits, lipoproteins comprising protein and lipids, etc. Specific binding partners may be naturally occurring substances but also substances prepared by means of, for example, chemical synthesis, microbiological techniques and/or genetic engineering processes. Examples which may be mentioned to illustrate but not restrict the term specific binding partner are: thyroxine binding globulin, steroid binding proteins, antibodies, antigens, haptens, enzymes, lectins, nucleic acids, repressors, oligonucleotides and polynucleotides, protein A, protein G, avidin, streptavidin, biotin, complement component C1q, nucleic acid binding proteins, etc. Specific binding pairs are for example: antibody/antigen, antibody/hapten, operator/repressor, nuclease/nucleotide, biotin/avidin, lectin/polysaccharide, steroid/steroid binding protein, active ingredient/active ingredient receptor, hormone/hormone receptor, enzyme/substrate, IgG/protein A, complementary oligonucleotides or polynucleotides, etc.
This invention also relates to the use of the polypeptides according to the invention in affinity chromatography, in particular for purifying specific binding partners binding to pCT.
The term xe2x80x9caffinity chromatographyxe2x80x9d means a method by which substances, in particular biopolymers, are purified and isolated and which is based on the fact that many substances can bind to their specific binding partners in a selective, noncovalent, reversible manner. The principle of the process involves the specific binding partner being bound in general covalently to an insoluble matrix (e.g. porous glasses, gels based on agarose, cellulose, dextran, polymer and silica gel) and brought into contact with a sample containing the substance. The sought-after substance is immobilized and retained by its specific interaction with the matrix-bound specific binding partner, while all other substances contained in the sample are removed by elution. The sought-after biopolymer is then detached from the matrix using a suitable eluent which cancels out the noncovalent bond between substance and specific binding partner (see also E. Buddecke (1989) Grundrisse der Biochemie, Walter de Gruyter, chapter 7 xe2x80x9cProteinexe2x80x9d).
This invention additionally relates to the use of the polypeptides according to the invention and/or the antibodies according to the invention as a diagnostic agent, as an ingredient of a diagnostic agent, for preparing a diagnostic agent, as a medicament, as an ingredient of a medicament and/or for preparing a medicament.
The term xe2x80x9cdiagnostic agentxe2x80x9d means in accordance with this invention an agent serving in particular to diagnose possible diseases, establish the state of health, the physical or mental state of organisms and/or detect or quantify substances or organisms in samples. In case of a xe2x80x9cin vitro diagnostic agentxe2x80x9d the analyte to be detected, for example procalcitonin or anti-procalcitonin antibodies, is detected in a sample outside a living human or animal organism and/or the concentration or amount thereof is determined. The polypeptides according to the invention and/or the antibodies according to the invention may also be administered to an organism as xe2x80x9cin vivo diagnostic agentsxe2x80x9d in unlabeled or labeled form using, for example, a radioactive isotope in the course of a function test or a scintigraphic method.
The polypeptides according to the invention may be processed further into, for example, physiologically active pCT cleavage products such as, in particular, calcitonin in order to prepare a medicament which may be used to influence calcium and bone metabolism. In addition, the polypeptides according to the invention themselves or else the antibodies according to the invention may be administered as medicaments either on their own or together with one or more pharmacologically active substances, for example for the treatment of tumors, sepsis and/or SIRS. The polypeptides according to the invention or else the antibodies according to the invention may also have their activity enhanced by modifications and/or linking to pharmacologically active substances.
This invention additionally comprises the polypeptides according to the invention and/or the antibodies according to the invention in a pharmaceutically suitable sterile injection medium. A pharmaceutically suitable sterile injection medium means, for example, a sterile pyrogen-free solution, for example saline or another electrolyte solution, such as is used conventionally in the intravenous, intramuscular, intraperitoneal or subcutaneous administration of medicaments, vaccines or contrast media.
The polypeptides according to the invention and/or the antibodies according to the invention may also be used in particular in a process for the quantitative or qualitative detection of an analyte, preferably procalcitonin or anti-procalcitonin antibodies, in a sample. In such a detection method according to the invention the polypeptides according to the invention may serve, for example, as standard antigen and/or specific binding partner in an analyte/binding partner complex.
In a quantitative test the amount or the concentration of the analyte in the sample is measured. The term xe2x80x9cquantitative testxe2x80x9d also comprises semi-quantitative methods which may measure only the approximate amount or concentration of the analyte in the sample or serve only to indicate relative quantities or concentrations. A qualitative test means detecting the presence of the analyte in the sample in fact or indicating that the analyte concentration in the sample is below or above a particular threshold or several particular thresholds.
The term xe2x80x9canalytexe2x80x9d means the substance to be detected. Examples of analytes are listed in EP-A2-0 515 194 on pages 8-15.
A xe2x80x9csamplexe2x80x9d in accordance with the invention means the material presumably containing the substance to be detected. The term sample comprises, for example, biological fluids or tissue, in particular of humans and animals such as blood, plasma, serum, sputum, exudate, bronchoalveolar lavage, lymph fluid, synovial fluid, seminal fluid, vaginal mucus, feces, urine, CSF, hairs, skin, tissue samples or tissue sections. Further comprised are cell culture samples, plant fluids or tissue, forensic samples, water and waste water samples, foods, medicaments. The samples need to be pretreated where appropriate to make the analyte available for the detection method or to remove interfering sample constituents. Such a pretreatment of samples may include removal and/or lysis of cells, precipitation, hydrolysis or denaturation of sample constituents such as, for example, proteins, centrifugation of samples, treatment of the sample using organic solvents such as, for example, alcohols, in particular methanol; treatment of the sample using detergents. Often the sample is transferred into another, usually aqueous, medium which, if possible, ought not to interfere with the assay.
The detection according to the invention of an analyte with the polypeptides according to the invention and/or the antibodies according to the invention may be carried out by methods such as, for example: Western blot, dot blot, immunohistochemical test methods, immunoelectrophoresis, immunofixation electrophoresis, electroimmunodiffusion, immunoprecipitation, radial immunodiffusion, immunofixation, immunochromatography, latex agglutination, turbidimetric or nephelometric test, homogeneous or heterogeneous binding assay, one- or two-step assay, sandwich assay, indirect assay, competitive assay, point-of-care tests, etc. These and other detection methods are described, for example, in xe2x80x9cLabor und Diagnosexe2x80x9d, ed. L. Thomas, TH-Books Verlagsgesellschaft mbH, Frankfurt, 1998, chapter 60, or in xe2x80x9cLaboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94An Introduction to Radioimmunoassay and Related Techniquesxe2x80x9d, ed. T. Chard, Elsevier, Amsterdam, 1987.
In binding assays the analyte, if present in the sample, is bound to one or more analyte-specific binding partners, and analyte/analyte-specific binding partner(s) complexes are formed.
In homogeneous binding assays free and complex-bound analytes are not separated. Examples of homogeneous immunoassays (see also Boguslaski and Li (1982) Applied Biochemistry and Biotechnology, 7:401-414) are many turbidimetric or nephelometric methods, it being possible for the specific binding partners used for the detection to be associated with latex particles; EMIT(copyright) assays; CEDIA(copyright) assays; fluorescence polarization immunoassays; luminescent oxygen channeling immunoassays (EP-A2-0 515 194; Ullman et al. (1994) Proc. Natl. Acad. Sci., 91:5426-5430; Ullman et al. (1996) Clinical Chemistry, 42:1518-1526); etc.
Heterogeneous binding assays comprise one or more separation steps and/or wash steps. The separation may be carried out by, for example, immunoprecipitation, precipitation using substances such as polyethylene glycol or ammonium sulfate, filtration, magnetic separation, binding to a solid phase such as, for example, to a test tube, a bead, a well of a microtiter plate or to filter paper or chromatography paper. In heterogeneous binding assays frequently one specific binding partner is associated with a reporter system component and one specific binding partner is associated with a solid phase (regarding indirect binding see also EP-A2-0 411 945). Here the specific binding partners may be different or the same, for example an analyte-specific monoclonal antibody may be employed both as capture agent (for example as a solid-phase antibody) and as labeled antibody if the analyte contains more than one epitope.
In heterogeneous sandwich assays the analyte is usually bound by a specific binding partner associated with a solid phase and a specific binding partner associated with a reporter system component. In the case of a sandwich immunoassay the specific binding partners may be analyte-specific antibodies or, if the analyte itself is an antibody, the antigen and/or a xe2x80x9cmodified antigenxe2x80x9d, for example a labeled antigen, antigen fragment, antigen analog. Examples of such sandwich complexes are: solid-phase antibody less than  greater than analyte less than  greater than antibody label or solid-phase antigen less than  greater than analyte (=antibody) less than  greater than antigen label.
A further embodiment of a heterogeneous immunoassay is the indirect immunoassay: in this case the analyte is an antibody. One of the specific binding partners is the antigen thereof and/or a modified antigen and the other specific binding partner is an antibody binding to the analyte and/or an immunoglobulin-binding protein. Examples of such complexes which may be formed in an indirect immunoassay are: solid phase anti-immunoglobulin antibody less than  greater than analyte (=anti-pCT antibody) less than  greater than labeled pCT or, alternatively, solid phase pCT less than  greater than analyte (=anti-pCT antibody) less than  greater than labeled protein A.
In a heterogeneous competitive immunoassay the sample analyte competes with a xe2x80x9cmodified analytexe2x80x9d, for example a labeled analyte, analyte fragment, analyte analog, etc. for a limited number of analyte-specific binding sites. Examples illustrating the principle are: (i) sample analyte competes with an analyte associated with a reporter system component for binding to a solid-phase associated analyte-specific antibody or (ii) sample analyte competes with a solid-phase associated analyte for binding to an analyte-specific antibody associated with a reporter system component.
Sandwich assays, competitive assays and indirect assays may also be carried out as homogeneous assay methods (see also EP-A2-0 515 194).
The term xe2x80x9cpoint-of-care testsxe2x80x9d or xe2x80x9cPOC testsxe2x80x9d has a broad meaning. It includes tests which do not need a separate analyzing or measuring device for carrying out or analysing the test. In many cases POC tests are based on immunochromatography methods, immune complex separations by filtration and/or immunofixation techniques. POC tests are intended in particular for on-the-spot measurements, for example at the hospital bed or at home, for the emergency doctor and/or the general practitioner and not so much for the large-scale laboratory. POC tests may also be carried out in particular by persons without in-depth training in medical technology and experience in the field of laboratory medicine. The term xe2x80x9cPOC testsxe2x80x9d in accordance with this invention also means so-called home tests or OTC (over the counter) tests which may be carried out by medical lay-persons such as, for example, the various pregnancy tests sold for home use. Further POC tests relate to, for example, the detection of heart attack markers, drugs, medicaments, infection markers and inflammation markers. In many POC tests specific binding partners are associated to filter or chromatography strips or disks during the course of the test. A positive or negative test reaction may be linked, for example, to the appearance or nonappearance of a colored band in a particular test field and/or the appearance or nonappearance of a particular symbol, for example xe2x80x9c+xe2x80x9d, xe2x80x9cxe2x88x92xe2x80x9d and/or the intensity of the respective measured signal.
A POC test for pCT, for example, may be constructed in the following way: the sample and labeled antibodies according to the invention which are capable of binding pCT are applied to a test strip. Suitable labels are, for example, colored latex particles, colloidal gold, enzymes, etc. If pCT is present in the sample, pCT/antibody complexes will be formed. These complexes move by means of capillary force toward a section where antibodies capable of binding to different pCT epitopes are fixed, for example as a band, or will be fixed during the course of the test (for example via a biotin/avidin bridge). The labeled PCT/antibody complexes are bound in this section and form a sandwich complex with the fixed antibodies. The intensity of the label signal is proportional to the pCT sample concentration in this case. In a competitive POC test method polypeptides according to the invention may be fixed, for example, in a section of the test strip or will be fixed in the course of the test. The fixed polypeptides according to the invention would compete with pCT from the sample for binding to labeled anti-pCT antibodies. Alternatively, fixed anti-pCT antibodies and labeled polypeptides according to the invention may also be employed for constructing a competitive pCT test.
A particularly preferred embodiment of the process according to the invention is a nephelometric or turbidimetric test, in particular a test which employs antibodies according to the invention and/or polypeptides according to the invention associated to latex particles. A further preferred process is a competitive assay in which the polypeptides according to the invention are associated to a solid phase and/or a reporter system component.
This invention further relates to polypeptides according to the invention associated to a solid phase and/or a reporter system component. In addition, the antibodies according to the invention may also be associated to a solid phase and or a reporter system component.
The term xe2x80x9cassociatedxe2x80x9d has a broad meaning and comprises, for example, covalent and noncovalent binding, direct and indirect binding, adsorption to a surface and enclosure in a depression or cavity, etc. In the case of covalent binding the polypeptides according to the invention and/or antibodies according to the invention are bound to the solid phase or label via a chemical bond. Examples of noncovalent binding are surface adsorption, enclosure in cavities or binding of two specific binding partners. Apart from directly binding to the solid phase or the label, the polypeptides according to the invention and/or the antibodies according to the invention may also be bound indirectly to the solid phase or the label through specific interaction with other specific binding partners (see also EP-A2-0 411 945). This is to be illustrated in more detail with examples: the biotinylated polypeptide according to the invention may be bound to the label via label-bound avidin; or a conjugate of fluorescein and polypeptide according to the invention may be bound to the solid phase via solid-phase bound anti-fluorescein antibodies; or the antibody according to the invention may be bound to the solid phase or the label via immunoglobulin-binding proteins.
The term xe2x80x9csolid phasexe2x80x9d in accordance with this invention comprises an object which consists of porous and/or non porous, generally water-insoluble material and which may have very diverse shapes such as, for example, vial, test tube, microtiter plate, bead, microparticle, stick, strip, filter paper or chromatography paper, etc. In general the surface of the solid phase is hydrophilic or may be made hydrophilic. The solid phase may comprise very diverse materials such as, for example, inorganic and/or organic materials, synthetic materials, naturally occurring materials and/or modified naturally occurring materials. Examples of solid-phase materials are polymers such as, for example, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; ceramics; glass; metals, in particular noble metals such as gold and silver; magnetite; mixtures or combinations thereof; etc. The term solid phase also comprises cells, liposomes or phospholipid vesicles.
The solid phase may have a coating of one or more layers of, for example, proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof in order to suppress or prevent, for example, nonspecific binding of sample constituents to the solid phase or to achieve, for example, improvements in suspension stability of particulate solid phases, shelf life, shaping stability or resistance to UV light, microbes or other destructive agents.
A xe2x80x9creporter systemxe2x80x9d may be one or more components, at least one component being a detectable label. A label means any molecule producing a signal by itself or capable of inducing production of a signal such as, for example, a fluorescent substance, radioactive substance, enzyme or chemiluminescent substance. The signal may be detected or measured, for example, by the enzyme activity, luminescence, light absorption, light scattering, electromagnetic or radioactive emission or a chemical reaction.
A label may be able to produce a detectable signal by itself so that no further components are necessary. Many organic molecules absorb ultraviolet and visible light, it being possible for these molecules to reach an excited energy level due to the energy transferred by light absorption and to emit the absorbed energy as light of a wavelength different from that of the incident light. Yet other labels may produce directly a detectable signal such as, for example, radioactive isotopes or dyes.
Yet other labels need additional components for signal production, that is to say the signal producing system in that case includes all components needed for generating the signal such as, for example, substrates, coenzymes, quenchers, accelerators, additional enzymes, substances reacting with enzyme products, catalysts, activators, cofactors, inhibitors, ions, etc.
Suitable labels (see also EP-A2-0 515 194; U.S. Pat. No. 5,340,716; U.S. Pat. No. 5,545,834; Bailey et al. (1987) J. Pharmaceutical and Biomedical Analysis 5:649-658) are, for example, enzymes including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, xcex2-galactosidase, luciferase, urease and acetylcholinesterase; dyes; fluorescent substances including fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonyl chloride and fluorescent rare earth chelates; chemiluminescent substances including luminol, isoluminol, acridine compounds, olefin, enol ether, enamine, aryl vinyl ether, dioxene, arylimidazole, lucigenin, luciferin and aequorin; sensitizers including eosin, 9,10-dibromoanthracene, methylene blue, porphyrin, phthalocyanin, chlorophyll, Rose Bengal; coenzymes; enzyme substrates; radioactive isotopes including 125I, 131I, 14C, 3H, 32P, 35S, 14C, 51Cr, 59Fe, 57Co and 75Se; particles including magnetic particles or particles, preferably latex particles, which may be labeled themselves by, for example, dyes, sensitizers, fluorescent substances, chemiluminescent substances, isotopes or other detectable labels; sol particles including gold and silver sols; liposomes or cells which may be labeled themselves by detectable labels; etc.
A reporter system may also comprise components which can interact with each other in a detectable manner at close range, for example as energy donors and energy acceptors such as, for example, photosensitizers and chemiluminescent substances (EP-A2-0 515 194), photosensitizers and fluorophores (WO 95/06877), radioactive iodine-125 and fluorophores (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82:8672-8676), fluorophores and fluorophores (Mathis (1993) Clin. Chem. 39:1953-1959) or fluorophores and fluorescence quenchers (U.S. Pat. No. 3,996,345).
An interaction between the components includes direct energy transfer between the components, for example by light or electron radiation and via short-lived reactive chemical molecules. It further comprises processes in which the activity of one component is inhibited or enhanced by one or more others, for example inhibition of or increase in enzyme activity or inhibition of, increase or change in electromagnetic radiation emitted from the affected component (e.g. wavelength shift, polarization). The interaction between the components also comprises enzyme cascades. In this case the components are enzymes, at least one of which supplies the substrate for a second one, resulting in maximum or minimum reaction velocity of the coupled substrate conversion.
An effective interaction between the components generally takes place when these are in spatial proximity, that is, for example, within a distance of a few xcexcm, in particular within a distance of below 600 nm, preferably below 400 nm, very particularly preferably below 200 nm.
Microparticles are commonly used as solid phase and/or label. The term xe2x80x9cmicroparticlexe2x80x9d in accordance with this invention means particles having an approximate diameter of at least 20 nm and no more than 20 xcexcm, usually between 40 nm and 10 xcexcm, preferably between 0.1 and 10 xcexcm, particularly preferably between 0.1 and 5 xcexcm, very particularly preferably between 0.15 and 2 xcexcm. The microparticles may be shaped regularly or irregularly. They may comprise spheres, spheroids, spheres having more or less large cavities or pores. The microparticles may comprise organic material, inorganic material or a mixture or combination of both. They may comprise porous or nonporous material, swellable or non-swellable material. In principle the microparticles may have any density, however, particles having a density close to the density of water such as from about 0.7 to about 1.5 g/ml are preferred. The preferred microparticles are suspendable in aqueous solutions and stable in suspension for as long as possible. They may be transparent, partly transparent or opaque. The microparticles may comprise a plurality of layers such as, for example, the so-called core-and-shell particles having a core and one or more surrounding layers. The term microparticle comprises, for example, dye crystals, metal sols, silica particles, glass particles, magnetic particles, polymer particles, oil drops, lipid particles, dextran, and protein aggregates. Preferred microparticles are particles suspendable in aqueous solutions and comprising water-insoluble polymer material, in particular substituted polyethylenes. Very particularly preferred are latex particles made of, for example, polystyrene, acrylic acid polymers, methacrylic acid polymers, acrylonitrile polymers, acrylonitrile/butadiene/styrene, polyvinyl acetate-acrylate, polyvinylpyridine, vinyl chloride/acrylate. Of particular interest are latex particles having on their surface reactive groups such as, for example, carboxyl, amino or aldehyde groups which facilitate covalent binding, for example of specific binding partners, to the latex particles. The preparation of latex particles is described in, for example, EP 0 080 614, EP 0 227 054 and EP 0 246 446.
The invention further relates to a test kit containing one or more of the antibodies according to the invention and/or one or more of the polypeptides according to the invention. Such a kit usually contains all or only some test components in packaged form. The antibodies according to the invention and the polypeptides according to the invention may be associated to, for example, one or more solid phases and/or one or more reporter system components. The test kit may contain, for example, standards; controls; and further reagents such as, for example, buffers, washing solutions, measured signal inducing solutions and/or enzyme substrate; cuvettes; pipettes and/or instructions. A particularly preferred test kit according to the invention contains polypeptides according to the invention and/or antibodies according to the invention associated to latex particles.
A particularly preferred embodiment of the invention is the use of the polypeptides according to the invention in standards and/or controls. In detection methods the concentration, amount or activity in a sample of a substance to be detected is commonly determined using reference or standard curves. In order to obtain such reference curves, standards, also called calibrators, containing a specific known concentration, amount or activity of the analyte are measured in the detection method. In the end, the concentration, amount or activity of the analyte in the sample can be determined by comparing the measured signal values of the sample with the reference curve.
Controls contain similarly to the standards a specific known concentration, amount or activity of the analyte or a modified analyte and serve to check the detection method.
To prepare the standards and/or controls according to the invention a specific amount of one or more of the polypeptides according to the invention is added to a matrix. This matrix may be, for example a human or animal serum or plasma or even an artificial matrix such as, for example, a buffer which may contain proteins and which may also contain further substances. Standards and controls may also contain one or more additional analytes. They can be in liquid, frozen or lyophilized form and therefore may be employed in the detection methods either directly or only after preparation.
The invention further relates to stable pCT solutions which may be used, for example, as controls, standards or else for other in vitro and in vivo applications. xe2x80x9cStablexe2x80x9d in this connection means that the desired property of the procalcitonin present in the solution, for example the ability to bind to specific antibodies remains generally unchanged over a specific period, in particular during liquid storage, while in xe2x80x9cunstablexe2x80x9d pCT solutions this property changes significantly over the same period.
Stable pCT solutions can be prepared by adding the polypeptides and sterol esters according to the invention to a serum/plasma-containing or serum/plasma-free matrix.
Particularly in pCT solutions employed as pCT controls and/or pCT standards the employed pCT may also be, for example, a peptide which has been isolated from natural sources or produced by recombination and which contains at least considerable parts of the amino acid sequence of human pCT such as, for example, relatively large pCT cleavage products. However, the peptide has to be suitable as standard and/or control serum antigen in a quantitative or qualitative pCT detection method.
The sterol esters suitable for preparing the pCT solution according to the invention belong to the steroid class of substances (gonane derivatives) which is generally characterized by an hydroxyl group in the 3 xcex2 position. The main differences are on the side chain located in the 17(20) position. The sterols constitute a large class (Beyer et al. (1981), Lehrbuch der organischen Chemie, pp 649-664 xe2x80x9cSteroidexe2x80x9d). Advantageously vitamin D3, estrone, stigmasterol and, particularly advantageously, cholesterol and derivatives thereof may be used.
The sterol esters to be employed according to the invention additionally and preferably have a polyethylene glycol group (PEG group) linked via a dicarboxylic acid. In principle all known dicarboxylic acids may be used; the use of succinic acid, adipic acid or sebacic acid is particularly advantageous.
In addition, the PEG group essentially ought to ensure the solubility of the sterol ester, so that the skilled worker is able to determine the optimal length easily, if necessary in an experiment. From experience the following chain lengths are advantageous: polyethylene glycol 600, polyethylene glycol 900 or polyethylene glycol 3000.
The sterol esters preferably employed for preparing the pCT solutions according to the invention are of the general formula I:
R1xe2x80x94Oxe2x80x94(O)Cxe2x80x94R2xe2x80x94C(O)xe2x80x94Oxe2x80x94[CH2xe2x80x94CH2xe2x80x94O]nxe2x80x94CH2xe2x80x94CH2xe2x80x94OHxe2x80x83xe2x80x83Formula I
where n=1-200 and
R1 is sterol,
R2 is an aliphatic or aromatic ring of 4 to 8 carbon atoms, wherein one or more of the ring atoms is optionally replaced by N, S or O, or is a linear aliphatic carbon chain having from 0 to 12 carbon atoms, or is a branched aliphatic carbon chain having from 0 to 12 carbon atoms, wherein 0 carbon atoms represents a bond; particularly preferred is the use of sterol esters in which R1 is a compound of the general formula II: 
xe2x80x83where R4 and R5 may be H or xe2x80x94CH3,
R6 is selected from a linear carbon chain having from 1 to 12 carbon atoms, a branched carbon chain having from 1 to 12 carbon atoms, an xe2x80x94OH group, and a xe2x95x90O group; and the rings A, B, C and D are each independently saturated, unsaturated or aromatic, and, if R4 is xe2x80x94C(19)H3, ring B may be opened between C(9) and C(10) with formation of a double bond between C(9) and C(19).
Very particularly preferred is the use of sterol esters, where the sterol residue originates from cholesterol, vitamin D3, stigmasterol or estrone. Advantageously the sterol ester is added in a concentration such that the concentration in the pCT solution is 0.05-5% by weight, preferably 0.1-3% by weight, particularly preferably 0.5-1.5% by weight.
The pCT solutions according to the invention may also contain protease inhibitors, for example aprotinin, benzamidine, bestatin, cystatin, pepstatin, PMSF, trypsin inhibitor, and/or detergents, in particular nonionic and/or zwitterionic detergents.
A particularly preferred embodiment of the pCT solution apart from the sterol esters also contains polygeline. Polygeline is a mixture of thermally degraded and cross-linked gelatin proteins and may be prepared according to DE-A 1155134 or U.S. Pat. No. 3,057,782. Advantageously polygeline is added such that the concentration in the pCT solution is 0.1-10% by weight, preferably 1-8% by weight, particularly preferably 2-6% by weight.
In order to measure the stabilizing effectxe2x80x94in particular in pCT solutions serving as standards and/or controlsxe2x80x94pCT may be determined, for example, in a nephelometric measurement method.
A further embodiment of this invention comprises the use of standards, controls and pCT solutions according to the invention in methods for quantitative or qualitative detection of pCT, calcitonin, katacalcin, N-procalcitonin and/or further pCT fragments in a sample.